Functional Characterization of Multiple Transactivating Elements in β-Catenin, Some of Which Interact with the TATA-binding Proteinin Vitro
1999; Elsevier BV; Volume: 274; Issue: 25 Linguagem: Inglês
10.1074/jbc.274.25.18017
ISSN1083-351X
AutoresAndreas Hecht, Claudia Litterst, Otmar Huber, Rolf Kemler,
Tópico(s)RNA Research and Splicing
Resumoβ-Catenin, a member of the family of Armadillo repeat proteins, plays a dual role in cadherin-mediated cell adhesion and in signaling by Wnt growth factors. Upon Wnt stimulation β-catenin undergoes nuclear translocation and serves as transcriptional coactivator of T cell factor DNA-binding proteins. Previously the transactivation potential of different portions of β-catenin has been demonstrated, but the precise location of transactivating elements has not been established. Also, the mechanism of transactivation by β-catenin and the molecular basis for functional differences between β-catenin and the closely related proteins Armadillo and Plakoglobin are poorly understood. Here we have used a yeast system for the detailed characterization of the transactivation properties of β-catenin. We show that its transactivation domains possess a modular structure, consist of multiple subelements that cover broad regions at its N and C termini, and extend considerably into the Armadillo repeat region. Compared with β-catenin the N termini of Plakoglobin and Armadillo have different transactivation capacities that may explain their distinct signaling properties. Furthermore, transactivating elements of β-catenin interact specifically and directly with the TATA-binding proteinin vitro providing further evidence that a major function of β-catenin during Wnt signaling is to recruit the basal transcription machinery to promoter regions of Wnt target genes. β-Catenin, a member of the family of Armadillo repeat proteins, plays a dual role in cadherin-mediated cell adhesion and in signaling by Wnt growth factors. Upon Wnt stimulation β-catenin undergoes nuclear translocation and serves as transcriptional coactivator of T cell factor DNA-binding proteins. Previously the transactivation potential of different portions of β-catenin has been demonstrated, but the precise location of transactivating elements has not been established. Also, the mechanism of transactivation by β-catenin and the molecular basis for functional differences between β-catenin and the closely related proteins Armadillo and Plakoglobin are poorly understood. Here we have used a yeast system for the detailed characterization of the transactivation properties of β-catenin. We show that its transactivation domains possess a modular structure, consist of multiple subelements that cover broad regions at its N and C termini, and extend considerably into the Armadillo repeat region. Compared with β-catenin the N termini of Plakoglobin and Armadillo have different transactivation capacities that may explain their distinct signaling properties. Furthermore, transactivating elements of β-catenin interact specifically and directly with the TATA-binding proteinin vitro providing further evidence that a major function of β-catenin during Wnt signaling is to recruit the basal transcription machinery to promoter regions of Wnt target genes. β-Catenin together with Plakoglobin and the more distantly related p120ctn (where ctn is catenin) belong to a large family of proteins that are involved in diverse cellular processes (1Peifer M. McCrea P.D. Green K.J. Wieschaus E. Gumbiner B.M. J. Cell Biol. 1992; 118: 681-691Crossref PubMed Scopus (308) Google Scholar). The members of this protein family are characterized by the presence of multiple copies of a 42-amino acid motif, the so-called Armadillo (Arm) 1The abbreviations used are: Arm, Armadillo; CBD, catenin-binding domain; DBD, DNA-binding domain; GST, glutathioneS-transferase; GTF, general transcription factor; LEF-1, lymphoid enhancer factor 1; PAGE, polyacrylamide gel electrophoresis; TAD, transactivation domain; TBP, TATA-binding protein; TCF, T cell factor; TLE, transducin-like enhancer of split; WT, wild type. repeat, which was named after a founding member of the family, the product of the Drosophila Armadillo gene (1Peifer M. McCrea P.D. Green K.J. Wieschaus E. Gumbiner B.M. J. Cell Biol. 1992; 118: 681-691Crossref PubMed Scopus (308) Google Scholar). In vertebrates, β-catenin and Plakoglobin were first described as components of cadherin-catenin cell adhesion complexes, where they link cadherin transmembrane proteins to α-catenin and the actin filaments of the cytoskeleton (2Kemler R. Trends Genet. 1993; 9: 317-321Abstract Full Text PDF PubMed Scopus (877) Google Scholar). Meanwhile, the dual involvement of β-catenin and its invertebrate homolog Armadillo in cell adhesion and cell-cell signaling is well established. These proteins are central components of the Wnt/wingless signal transduction cascade which for example functions to specify anterior-posterior segment polarity inDrosophila larvae or to determine the embryonic dorso-anterior body axes in Xenopus laevis (3Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2228) Google Scholar, 4Moon R.T. Brown J.D. Torres M. Trends Genet. 1997; 13: 157-162Abstract Full Text PDF PubMed Scopus (546) Google Scholar, 5Willert K. Nusse R. Curr. Opin. Genet. & Dev. 1998; 8: 95-102Crossref PubMed Scopus (666) Google Scholar). Wnt/wingless growth factors are secreted glycoproteins that utilize members of the Frizzled family of seven transmembrane domain proteins as receptors (5Willert K. Nusse R. Curr. Opin. Genet. & Dev. 1998; 8: 95-102Crossref PubMed Scopus (666) Google Scholar, 6Bhanot P. Brink M. Samos C.H. Hsieh J.C. Wang Y. Macke J.P. Andrew D. Nathans J. Nusse R. Nature. 1996; 382: 225-230Crossref PubMed Scopus (1228) Google Scholar). Stimulation of Frizzled receptors by Wnt activates a signaling pathway which includes Dishevelled, GSK-3β, Axin, or Conductin and the adenomatous polyposis coli tumor suppressor protein. GSK-3β, Axin, and adenomatous polyposis coli are thought to form a multiprotein complex that regulates the stability of β-catenin and thereby its subcellular distribution (5Willert K. Nusse R. Curr. Opin. Genet. & Dev. 1998; 8: 95-102Crossref PubMed Scopus (666) Google Scholar, 7Ben-Ze'ev A. Geiger B. Curr. Opin. Cell Biol. 1998; 10: 629-639Crossref PubMed Scopus (307) Google Scholar). In cells receiving a Wnt signal β-catenin is translocated into the nucleus where it interacts with transcription factors of the TCF family (5Willert K. Nusse R. Curr. Opin. Genet. & Dev. 1998; 8: 95-102Crossref PubMed Scopus (666) Google Scholar, 7Ben-Ze'ev A. Geiger B. Curr. Opin. Cell Biol. 1998; 10: 629-639Crossref PubMed Scopus (307) Google Scholar, 8Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2595) Google Scholar, 9Huber O. Korn R. McLaughlin J. Ohsugi M. Herrmann B.G. Kemler R. Mech. Dev. 1996; 59: 3-10Crossref PubMed Scopus (782) Google Scholar, 10Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destree O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1619) Google Scholar, 11Cavallo R. Rubenstein D. Peifer M. Curr. Opin. Genet. & Dev. 1997; 7: 459-466Crossref PubMed Scopus (78) Google Scholar, 12Clevers H. van de Wetering M. Trends Genet. 1997; 13: 485-489Abstract Full Text PDF PubMed Scopus (254) Google Scholar). Formation of β-catenin-TCF complexes ultimately leads to the activation of specific target genes such as Ultrabithorax,siamois, twin, nodal-related-3 or c-MYC (13Brannon M. Gomperts M. Sumoy L. Moon R.T. Kimelman D. Genes Dev. 1997; 11: 2359-2370Crossref PubMed Scopus (466) Google Scholar, 14McKendry R. Hsu S.C. Harland R.M. Grosschedl R. Dev. Biol. 1997; 192: 420-431Crossref PubMed Scopus (213) Google Scholar, 15Riese J. Yu X. Munnerlyn A. Eresh S. Hsu S.C. Grosschedl R. Bienz M. Cell. 1997; 88: 777-787Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 16He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4084) Google Scholar, 17Laurent M.N. Blitz I.L. Hashimoto C. Rothbacher U. Cho K.W. Development. 1997; 124: 4905-4916Crossref PubMed Google Scholar). Whereas the importance of the β-catenin-TCF complex during activation of Wnt target genes is well established, the specific function of β-catenin and its mechanism of action are less clear. TCF factors are sequence-specific DNA-binding proteins with a high mobility group domain recognizing a common consensus motif but which lack classical transactivation domains (12Clevers H. van de Wetering M. Trends Genet. 1997; 13: 485-489Abstract Full Text PDF PubMed Scopus (254) Google Scholar, 18Grosschedl R. Giese K. Pagel J. Trends Genet. 1994; 10: 94-100Abstract Full Text PDF PubMed Scopus (734) Google Scholar). Instead, LEF-1, for instance, performs architectural functions or relies on interactions with accessory factors such as TLE/groucho co-repressors or a transcriptional coactivator termed ALY to modulate gene expression (19Levanon D. Goldstein R.E. Bernstein Y. Tang H. Goldenberg D. Stifani S. Paroush Z. Groner Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11590-11595Crossref PubMed Scopus (410) Google Scholar, 20Roose J. Molenaar M. Peterson J. Hurenkamp J. Brantjes H. Moerer P. van de Wetering M. Destree O. Clevers H. Nature. 1998; 395: 608-612Crossref PubMed Scopus (576) Google Scholar, 21Bruhn L. Munnerlyn A. Grosschedl R. Genes Dev. 1997; 11: 640-653Crossref PubMed Scopus (255) Google Scholar). β-Catenin could therefore act either by inducing changes in promoter structure (8Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2595) Google Scholar), by alleviating repression through displacement of the TLE/groucho factors, or by providing a TAD. In fact, a TAD has been identified at the C terminus of β-catenin (22van de Wetering M. Cavallo R. Dooijes D. van Beest M. van Es J. Loureiro J. Ypma A. Hursh D. Jones T. Bejsovec A. Peifer M. Mortin M. Clevers H. Cell. 1997; 88: 789-799Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar). This domain is both necessary and sufficient for the signaling activity of β-catenin in early Xenopus development (23Vleminckx K. Kemler R. Hecht A. Mech. Dev. 1999; 81: 49-58Crossref Scopus (89) Google Scholar), and in the fly the absence of the corresponding region from Armadillo causes diverse developmental defects (24Klingensmith J. Noll E. Perrimon N. Dev. Biol. 1989; 134: 130-145Crossref PubMed Scopus (51) Google Scholar, 25Peifer M. Wieschaus E. Cell. 1990; 63: 1167-1176Abstract Full Text PDF PubMed Scopus (385) Google Scholar). Thus, the currently prevailing view is that docking of β-catenin to TCF factors primarily serves to deliver the C-terminal TAD to promoter elements of Wnt target genes and thereby to attract the basal transcription machinery (5Willert K. Nusse R. Curr. Opin. Genet. & Dev. 1998; 8: 95-102Crossref PubMed Scopus (666) Google Scholar, 12Clevers H. van de Wetering M. Trends Genet. 1997; 13: 485-489Abstract Full Text PDF PubMed Scopus (254) Google Scholar, 22van de Wetering M. Cavallo R. Dooijes D. van Beest M. van Es J. Loureiro J. Ypma A. Hursh D. Jones T. Bejsovec A. Peifer M. Mortin M. Clevers H. Cell. 1997; 88: 789-799Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar). Therefore, it is important to seek evidence for an interaction between β-catenin and the general transcription apparatus. β-Catenin shares 68 and 71% sequence similarity with Plakoglobin and Armadillo, respectively (1Peifer M. McCrea P.D. Green K.J. Wieschaus E. Gumbiner B.M. J. Cell Biol. 1992; 118: 681-691Crossref PubMed Scopus (308) Google Scholar). All three proteins can interact with TCFs (7Ben-Ze'ev A. Geiger B. Curr. Opin. Cell Biol. 1998; 10: 629-639Crossref PubMed Scopus (307) Google Scholar, 9Huber O. Korn R. McLaughlin J. Ohsugi M. Herrmann B.G. Kemler R. Mech. Dev. 1996; 59: 3-10Crossref PubMed Scopus (782) Google Scholar) and possess transactivation domains at their C termini (22van de Wetering M. Cavallo R. Dooijes D. van Beest M. van Es J. Loureiro J. Ypma A. Hursh D. Jones T. Bejsovec A. Peifer M. Mortin M. Clevers H. Cell. 1997; 88: 789-799Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar, 26Simcha I. Shtutman M. Salomon D. Zhurinsky J. Sadot E. Geiger B. Ben-Ze'ev A. J. Cell Biol. 1998; 141: 1433-1448Crossref PubMed Scopus (239) Google Scholar). Nonetheless neither β-catenin nor Plakoglobin can rescue the signaling defects of Armadillo mutant flies (27White P. Aberle H. Vincent J.P. J. Cell Biol. 1998; 140: 183-195Crossref PubMed Scopus (52) Google Scholar), even though β-catenin can interact with Pangolin/dLEF-1 and murine LEF-1 can function as a downstream effector of Armadillo inDrosophila (15Riese J. Yu X. Munnerlyn A. Eresh S. Hsu S.C. Grosschedl R. Bienz M. Cell. 1997; 88: 777-787Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 28Brunner E. Peter O. Schweizer L. Basler K. Nature. 1997; 385: 829-833Crossref PubMed Scopus (447) Google Scholar). Moreover, Plakoglobin cannot replace β-catenin in mouse embryonic development (29Haegel H. Larue L. Ohsugi M. Fedorov L. Herrenknecht K. Kemler R. Development. 1995; 121: 3529-3537Crossref PubMed Google Scholar), and it is dispensable during early Xenopus development, whereas β-catenin is absolutely required to establish Spemann organizer activity (30Kofron M. Spagnuolo A. Klymkowsky M. Wylie C. Heasman J. Development. 1997; 124: 1553-1560PubMed Google Scholar, 31Heasman J. Crawford A. Goldstone K. Garner-Hamrick P. Gumbiner B. McCrea P. Kintner C. Noro C.Y. Wylie C. Cell. 1994; 79: 791-803Abstract Full Text PDF PubMed Scopus (590) Google Scholar). Although the basis for these differences is unknown, it is likely that they are associated with β-catenin, Plakoglobin, and Armadillo themselves, and further characterization of their transactivation properties might provide insight into their distinct functions. To understand better the signaling activities of β-catenin and its relatives, we have systematically investigated their transactivation properties. Since analyses of catenin-dependent transcriptional activation in vertebrate cells is frequently biased by the presence of endogenous β-catenin (26Simcha I. Shtutman M. Salomon D. Zhurinsky J. Sadot E. Geiger B. Ben-Ze'ev A. J. Cell Biol. 1998; 141: 1433-1448Crossref PubMed Scopus (239) Google Scholar, 32Merriam J.M. Rubenstein A.B. Klymkowsky M.W. Dev. Biol. 1997; 185: 67-81Crossref PubMed Scopus (62) Google Scholar, 33Miller J.R. Moon R.T. J. Cell Biol. 1997; 139: 229-243Crossref PubMed Scopus (157) Google Scholar), we have used the yeast Saccharomyces cerevisiae as a model system that does not possess cadherins, catenins, TCFs, or homologs of any of the components of the Wnt signaling pathway except GSK-3β. Our results indicate that the overall activity of β-catenin stems from the functional cooperation of multiple transactivating elements distributed over broad regions at both its C terminus and N terminus, as well as parts of the Arm repeat region. Interestingly, β-catenin differs from Plakoglobin and Armadillo with respect to the transactivation capacities of its N terminus. This could, at least in part, explain their distinct signaling characteristics. We also show that transactivating elements of β-catenin can specifically interact with the TATA-binding protein in vitro. This further supports the model that a major function of β-catenin during Wnt signaling is to recruit components of the basal transcription machinery to promoter regions of β-catenin/TCF target genes. The strains used in this study were EGY48 (MATa, his3, trp1, ura3-1, leu2::pLEU2-LexAop6) (34Finley J.R. Brent R. Glover D.M. Hames B.D. DNA Cloning: A Practical Approach. Oxford University Press, Oxford, UK1995: 169-203Google Scholar) and its derivative AYH50 that carries a single chromosomal copy of the LexAop8-Gal1-lacZ::URA3 cassette in the ura3locus. Yeast were grown under standard conditions in rich or synthetic minimal media (35Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar). All plasmids were constructed and purified using standard procedures (36Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Sequences of plasmids generated with polymerase chain reaction fragments were verified using the BigDye dideoxy cycle sequencing kit (Applied Biosystems) on an Applied Biosystems 310 sequencer. The complete coding regions for mouse β-catenin from pGEX4T1MMBC (37Aberle H. Butz S. Stappert J. Weissig H. Kemler R. Hoschuetzky H. J. Cell Sci. 1994; 107: 3655-3663Crossref PubMed Google Scholar), human Plakoglobin from pGEX4T1HPG (37Aberle H. Butz S. Stappert J. Weissig H. Kemler R. Hoschuetzky H. J. Cell Sci. 1994; 107: 3655-3663Crossref PubMed Google Scholar), and human p120ctn from pGEXp120 (38Geis K. Aberle H. Kuhl M. Kemler R. Wedlich D. Dev. Genes & Evolut. 1998; 207: 471-481Crossref PubMed Scopus (30) Google Scholar) were inserted, respectively, into the SmaI/XhoI sites,NcoI/EcoRI sites, and EcoRI site of p570.1, which was made by cloning a ScaI/SalI fragment carrying the ADH1 promoter/terminator cassette from pACT2 (CLONTECH) without the GAL4 activation domain, into pRS424 (39Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene (Amst.). 1992; 110: 119-122Crossref PubMed Scopus (1434) Google Scholar) cut with ScaI and XhoI. The parental plasmid for expression of the LexA fusions was p573.2, which is a derivative of pEG202 (34Finley J.R. Brent R. Glover D.M. Hames B.D. DNA Cloning: A Practical Approach. Oxford University Press, Oxford, UK1995: 169-203Google Scholar) based on the pRS423 backbone (39Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene (Amst.). 1992; 110: 119-122Crossref PubMed Scopus (1434) Google Scholar). To generate the LexA-LEF-1 expression vector, the entire coding region of murine LEF-1 (9Huber O. Korn R. McLaughlin J. Ohsugi M. Herrmann B.G. Kemler R. Mech. Dev. 1996; 59: 3-10Crossref PubMed Scopus (782) Google Scholar) was cloned into p573.2. From this construct anEcoRI/EcoNI fragment or anEcoNI/XhoI fragment was deleted to obtain LexA-LEFΔN or LexA-LEFΔC. To make constructs coding for LexA-β-catenin, LexA-p120ctn, LexA-Plakoglobin, and LexA-Armadillo, an EcoRI/BamHI fragment from pGEX4T1MMBC, a NcoI/XhoI fragment from pGEX4T1HPG, an EcoRI fragment from pGEXp120, or aBamHI/NotI fragment from pGEXArmadillo was inserted into p573.2. Mutants with N-terminal deletions of LexA-β-catenin, LexA-Plakoglobin, and LexA-Armadillo were generated by in-frame insertion of suitable restriction fragments into p573.2. C-terminal deletions of LexA-β-catenin, LexA-Plakoglobin, and LexA-Armadillo were obtained by cutting of the corresponding full-length constructs with appropriate restriction enzymes within coding regions and downstream thereof and religating. Deletion constructs for fine mapping of transactivating elements in β-catenin, Plakoglobin, and Armadillo were made by polymerase chain reaction amplification of the desired fragments and insertion into theEcoRI/BamHI sites of p573.2. To obtain plasmids for the expression of GAL4-β-catenin fusion proteins in mammalian cells, we excised EcoRI/NotI restriction fragments coding for the desired β-catenin portions from the appropriate yeast expression vectors and cloned them into theEcoRI/Bsp120I sites downstream of the GAL4 DBD in pCMVGAL4 (40Mink S. Haenig B. Klempnauer K.H. Mol. Cell. Biol. 1997; 17: 6609-6617Crossref PubMed Google Scholar). The luciferase reporter construct pG5E1bLuc was made by inserting a PvuII/BamHI fragment from pG5E1bCAT (41Lillie J.W. Green M.R. Nature. 1989; 338: 39-44Crossref PubMed Scopus (471) Google Scholar) into the SmaI/BglII sites of pGL3Basic (Promega). Expression plasmids for GST fusions with β-catenin residues 1–781, 1–284, 1–183, 1–119, or 683–781 and WT or histidine-tagged human TBP (TBP-His6) have been described (37Aberle H. Butz S. Stappert J. Weissig H. Kemler R. Hoschuetzky H. J. Cell Sci. 1994; 107: 3655-3663Crossref PubMed Google Scholar, 42Bauer A. Huber O. Kemler R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14787-14792Crossref PubMed Scopus (170) Google Scholar). All other GST fusions were generated by inserting appropriate DNA fragments from the LexA-expression plasmids or by inserting polymerase chain reaction-derived DNA fragments into theEcoRI/XhoI sites of pGEX-2TK with the polylinker region from pGEX-4T-1 (Amersham Pharmacia Biotech). Yeast strains were transformed by the lithium acetate method (43Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2895) Google Scholar) and plated on appropriate selective media. To determine β-galactosidase activity in EGY48, individual transformants were pregrown to saturation in selective media, diluted into YEPD to an A600 of 0.2, and regrown until mid-log phase. One ml from each culture was transferred into a 1.5-ml Eppendorf tube. Cells were pelleted, washed once in 1 ml of Z buffer (0.1 m sodium phosphate, pH 7.0, 10 mm KCl, 1 mm MgSO4), resuspended in 200 μl of Z buffer, and permeabilized by two cycles of freezing and thawing. Of the final cell suspension 20 μl was used to determine β-galactosidase activity as described (23Vleminckx K. Kemler R. Hecht A. Mech. Dev. 1999; 81: 49-58Crossref Scopus (89) Google Scholar). Values given represent the average from at least three independent measurements with several isolates for each plasmid combination and are expressed as relative light units (RLU) per A 600 of cells. To determine β-galactosidase activity after transformation of AYH50, samples corresponding to one-tenth of the transformation reaction were grown to saturation in selective media. The cultures were then diluted to an A 600 0.2 and analyzed for β-galactosidase activity as above. Each measurement from these pool cultures averages the activity of approximately 1000 independent primary transformants. For preparation of whole cell extracts the same cultures were used as for determining β-galactosidase activity. From each culture aliquots with 2 A 600 units of cells were pelleted, washed once in sterile water, and resuspended in 50 μl of 50 mm HEPES/NaOH, pH 7.4, 300 mm NaCl, 1 mm EDTA, 10% glycerol, 0.5% Nonidet P-40, and 0.4% "Complete" protease inhibitor mix (Roche Molecular Biochemicals). An equal volume of glass beads was added, and cell lysis was carried out by vortexing for 5 min on an IKA Vibrax VXR mixer at 4 °C. After addition of SDS-PAGE loading buffer 0.2 A 600units were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose. Western blotting experiments were performed as described (23Vleminckx K. Kemler R. Hecht A. Mech. Dev. 1999; 81: 49-58Crossref Scopus (89) Google Scholar) using polyclonal antisera against LexA (Invitrogen) or mouse monoclonal antibodies against β-catenin, Plakoglobin, and p120ctn (Transduction Laboratories). Human 293 kidney cells (ATCC number CRL-1573) were transfected by the calcium-phosphate coprecipitation method as described (23Vleminckx K. Kemler R. Hecht A. Mech. Dev. 1999; 81: 49-58Crossref Scopus (89) Google Scholar) using 1.0 μg of luciferase reporter plasmid, 0.5 μg of expression vector for the GAL4-β-catenin fusions, and 1.0 μg of pRSVLacZ as internal control. Cell lysis occurred in 100 μl of 50 mm Tris phosphate, pH 7.8, 250 mm KCl, 0.1% Nonidet P-40, 10% glycerol, on ice for 20 min. Luciferase activities were determined as before (23Vleminckx K. Kemler R. Hecht A. Mech. Dev. 1999; 81: 49-58Crossref Scopus (89) Google Scholar) and normalized against β-galactosidase activities. Average values from at least three independent transfection experiments are given. Nuclear extracts from HeLa cells grown to confluence in eight 15-cm tissue culture dishes were prepared as described (44Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar) except that nuclei were extracted in 1 volume of elution buffer (20 mm HEPES/KOH, pH 7.5, 420 mm NaCl, 25% glycerol, 1.5 mmMgCl2, 0.2 mm EDTA, 0.5 mmdithiothreitol, and Complete protease inhibitor mix). After elution the nuclei were removed by centrifugation at 25,000 × gfor 30 min, and the nuclear extract was stored in small aliquots at −80 °C without dialysis. For use in pull-down assays the crude nuclear extracts were diluted 1:5 in elution buffer without NaCl. GST fusion proteins were expressed in Escherichia coli BL21 (DE3) and purified with GSH-Sepharose beads (Amersham Pharmacia Biotech) as described (9Huber O. Korn R. McLaughlin J. Ohsugi M. Herrmann B.G. Kemler R. Mech. Dev. 1996; 59: 3-10Crossref PubMed Scopus (782) Google Scholar, 37Aberle H. Butz S. Stappert J. Weissig H. Kemler R. Hoschuetzky H. J. Cell Sci. 1994; 107: 3655-3663Crossref PubMed Google Scholar). After elution from the GSH-Sepharose fusion proteins were dialyzed against 50 mm Tris/HCl, pH 8.0, concentrated using Centricon ultrafiltration cartridges where necessary, frozen in liquid nitrogen, and stored at −80 °C until use. To express TBP-His6 in E. coli the plasmid pQE60-TBP was transformed into E. coli M15/pREP4 (Qiagen) and TBP-His6 was purified by ion exchange and metal affinity chromatography as described (42Bauer A. Huber O. Kemler R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14787-14792Crossref PubMed Scopus (170) Google Scholar, 45Kato K. Makino Y. Kishimoto T. Yamauchi J. Kato S. Muramatsu M. Tamura T. Nucleic Acids Res. 1994; 22: 1179-1185Crossref PubMed Scopus (45) Google Scholar). Approximately 10 μl bed volume of GSH-Sepharose beads (Amersham Pharmacia Biotech) was preincubated in 200 μl of pull-down buffer (20 mm HEPES/KOH, pH 7.5, 100 mm KCl, 5 mm MgCl2, 0.5 mm EDTA, 0.05% Nonidet P-40, 1 mmdithiothreitol, 0.02% bovine serum albumin, and Complete protease inhibitor) mix together with the various GST fusion proteins. For pull-down assays from nuclear extracts 5 μg GST or GST fusion protein was used; for pull-down assays from reticulocyte lysates we used 1 μg of GST or GST fusion proteins with C-terminal portions of β-catenin and 5 μg of GST or GST fusion proteins with N-terminal portions of β-catenin, respectively. After 30 min of preincubation binding reactions were supplemented with 20 μl of the Ni-NTA eluate containing approximately 500 ng of TBP-His6 or with 1 μl of reticulocyte lysate containing human TBP that had been transcribed from pCS2+TBP and translated in vitro in the presence of [35S]methionine as described by the manufacturer (SP6 TNT system, Promega). For pull-down experiments from nuclear extracts, the prebinding solution was removed and replaced with 500 μl of diluted nuclear extract (approximately 500 μg of protein) from HeLa cells. Binding reactions were done on a shaker platform for 2 h at 4 °C. The GSH-Sepharose beads were pelleted; the supernatant with unbound material was removed; and the beads were washed extensively in pull-down buffer with 200 mm KCl. Bound proteins were eluted from the GSH beads with SDS-PAGE loading buffer, resolved by electrophoresis on 10% SDS-polyacrylamide gels, and analyzed by Western blotting with mouse monoclonal anti-TBP antibodies (Transduction Laboratories, Promega) or by fluorography after soaking the gels in Amplify solution (Amersham Pharmacia Biotech). To determine whether it was possible to analyze transactivation by β-catenin and other Arm family members in the yeast S. cerevisiae, we inserted the coding regions for β-catenin, Plakoglobin, and p120ctn into a multicopy yeast expression vector. Full-length LEF-1, the N-terminal CBD of LEF-1 (8Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2595) Google Scholar, 9Huber O. Korn R. McLaughlin J. Ohsugi M. Herrmann B.G. Kemler R. Mech. Dev. 1996; 59: 3-10Crossref PubMed Scopus (782) Google Scholar), or a C-terminal portion lacking the CBD (Fig.1 A) were fused to the LexA DNA-binding domain and also inserted into a multicopy yeast expression plasmid. Combinations of the resulting plasmids were transformed into the yeast strain EGY48 carrying a plasmid-borne lacZreporter gene driven by the GAL1 minimal promoter and the LexA operator (34Finley J.R. Brent R. Glover D.M. Hames B.D. DNA Cloning: A Practical Approach. Oxford University Press, Oxford, UK1995: 169-203Google Scholar). Individual transformants were isolated and analyzed for expression of the LexA-LEF-1 fusion proteins, β-catenin, Plakoglobin, and p120ctn. All factors were properly expressed as shown by Western blotting experiments performed on whole cell extracts that were probed with anti-LexA antibodies to detect the different LexA-LEF-1 fusions (Fig. 1 B, lanes 1–4) or monoclonal antibodies against β-catenin, Plakoglobin, and p120ctn(Fig. 1 B, lanes 5–13). Next, we analyzed expression of the lacZ gene (Fig.1 C). As expected, no activation of the lacZreporter gene was seen in control experiments with the catenins alone (Fig. 1 C, lines 1–4) or with the LexA-LEF-1 fusion proteins in the absence of the catenins (Fig. 1 C, lines 5, 9 and12). Also, the combination of LexA-LEF-1 and p120ctn had no effect on reporter gene activity (Fig.1 C, line 8). In contrast, coexpression of β-catenin or Plakoglobin and LexA-LEF-1 strongly activated the lacZ gene (Fig. 1 C, lines 6 and 7). The addition of the LEF-1 CBD to the LexA DNA-binding domain was sufficient to allow activation of the lacZ gene by β-catenin or Plakoglobin, whereas no transactivation was seen with LexA-LEFΔN lacking the CBD (Fig. 1 C, lines 10, 11, 13, and 14). Thus, as in vertebrate cells, β-catenin and Plakoglobin can interact with LEF-1 through the CBD and function as transcriptional coactivators of LEF-1 when expressed in yeast. B
Referência(s)